The interplay between cationic peptide sequence, siRNA and electrolyte: formulating lipopolyplexes in NaCl solution affords nanocomplexes with exceptional levels of gene silencing

Abstract

Lipopolyplexes are important non-viral vectors for the delivery of therapeutic siRNA. However, many formulations developed are unstable or show poor cellular uptake under physiologically relevant conditions, for example in the presence of serum. In this work, we have studied the effects of formulating ternary lipopolyplexes in NaCl solution on nanocomplex structure and gene silencing activity. The lipopolyplexes were prepared from vesicles containing a 1 : 1 mixture of the cationic lipid DOTMA and the helper lipid DOPE, co-formulated with singly branched cationic peptides attached to a targeting sequence and with siRNA, in sodium chloride solution. These lipopolyplexes retained high levels of gene silencing in the presence of media supplemented with fetal bovine serum. Exceptionally good activity was observed when His- or His-Arg cationic sequences were used, superior to control formulations with Lipofectamine 2000. We have shown that these lipopolyplexes are large, multilamellar complexes which efficiently encapsulate and protect siRNA. In addition, the most effective His- or His-Arg cationic sequences showed looser complexation between the cationic peptide and the siRNA. This work thus represents a significant advance in the optimisation of lipopolyplex formulations for therapeutic gene silencing in vivo under physiologically relevant conditions, paving the way for more effective and less toxic siRNA-based therapies for clinical use.